Biomedical subjects

T Lamla

Publications and source records attributed to T Lamla.

3 recordsLinked to original sources

PubMed2006

Screening for soluble expression constructs using cell-free protein synthesis.

The SH2 domain of STAT6 was chosen to test the in vitro protein synthesis as a screening tool. Goal of the screening was to obtain constructs which produce soluble protein in E. coli. The expression of 70 different constructs using an E. coli based cell-free system revealed two constructs, which give partly soluble protein. The introduction of two mutations, which had been suggested by a structural based alignment of 20 different SH2 domains lead to increased solubility. The expression of both constructs in E. coli followed by an affinity and size exclusion chromatography resulted in milligram quantities of highly purified protein.

Animals↗

PubMed2001

In vitro selection of other proteins than antibodies by means of ribosome display.

The in vitro selection and simultaneous evolution of proteins is feasible by means of ribosome display. Here, we describe the use of a protein bearing a binding property without being an antibody for affinity enrichment of the ternary complex, consisting of a protein, a ribosome and a encoding mRNA. The binding property was a simple affinity tag, namely Strep-tag II and His-tag. We could demonstrate that the selection of a specific mRNA encoding a shortened bovine heart fatty acid binding protein with a N-terminal His-tag was possible. After nine cycles of transcription, translation, affinity selection and reverse transcription PCR the protein with the His-tag could be enriched 10(8)-fold.

Animals↗

PubMed2001

The cell-free protein biosynthesis--applications and analysis of the system.

The in vitro protein biosynthesis has the potentials to become a powerful technology for biochemical research. Beside the determination of structure and function the in vitro evolution of proteins is also of great interest. The system described was used to produce bovine heart fatty acid binding protein (FABP) and bacterial chloramphenicol acetyltransferase (CAT) with and without fusion of the Strep-tag II affinity peptide. The proteins were purified after and during protein biosynthesis by using a StrepTactin Sepharose matrix. No significant influence of the Strep-tag and the conditions during the affinity chromatography on maturation or activity of the protein was observed. The in vitro evolution of proteins is feasible by means of ribosome display. The selection of a specific mRNA coding for a shortened FABP with a N-terminal His-tag via the accompanying protein property was shown. Goal of the selection was to bind the FABP via the His-tag on Ni(II)-IDA-agarose. After nine cycles of transcription, translation, affinity selection and RT-PCR the protein with the His-tag could be enriched 10(8)-fold. In order to correlate a possible relationship between changes in protein population and biological function studies were initiated in which 2-dimensional protein patterns of the total in vitro system were compared after 0 and 2 h reaction time. The very interesting findings are that a number of proteins disappear, while others are newly formed during protein synthesis.

Animals↗